Colocalization of Rab27b with α and dense granules in MKs. (A-B) Immunolocalization of Rab27b in the MK cell body (A) and in proplatelet extensions (B). Staining is granular in both sites. MKs were cytocentrifuged onto glass slides, fixed, and stained with the Rab27b-specific R27B1 antibody, followed by FITC-conjugated goat antirabbit IgG. (C) Rab27b is not exclusively associated with the lineage-specific thick marginal microtubule cytoskeleton, as revealed by costaining with R27B1 and a mouse β-tubulin monoclonal antibody (detected using Texas Red-conjugated donkey antimouse IgG). (D-I) Colocalization of Rab27b with the α-granule marker VWF in the cell body and in proplatelet shafts, as revealed through separate immunostaining for Rab27b (D,G; antibody directly conjugated to FITC), VWF (E,H; antibody directly conjugated to Texas Red), and merging of the images (F,I). (J-L) Colocalization of Rab27b with the dense-granule marker serotonin in proplatelets, as revealed through separate immunostaining for Rab27b (J), serotonin (K; rat antiserotonin detected using Texas Red-conjugated goat antirat IgG), and merging of the images (L). Original magnifications, × 600 (A, D-F); and × 1000 (B-C, G-L).